Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 μM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy.

中文翻译：

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硫醇稳态的作用机制和对经典高胱氨酸尿症酶替代疗法疗效的影响。


胱硫醚 β-合酶 （CBS） 缺乏引起的高胱氨酸尿症 （HCU） 的特征是血浆和组织同型半胱氨酸水平升高。HCU 无法治愈，但通常通过限制蛋氨酸/蛋白质和补充维生素 B6 来控制 HCU。基于人 CBS 的酶替代疗法 （ERT） 已经开发出来，并通过使血浆总同型半胱氨酸低于临床相关的 100 μM 阈值，在几种小鼠模型中显示出纠正 HCU 表型的显著疗效。由于同型半胱氨酸的反应性促进二硫键形成和蛋白质结合，并且 ERT 无法使血浆总同型半胱氨酸水平正常化，因此 ERT 在 HCU 中的作用机制仍有待进一步表征。在这里，我们表明只有还原的同型半胱氨酸作为 CBS 的底物，其可用性限制了 CBS 的同型半胱氨酸降解能力。我们还证明，细胞以还原形式输出同型半胱氨酸，其在培养基中被 CBS 有效代谢。丝氨酸（一种 CBS 共底物）的可用性并不是我们基于细胞的模型中的限制因素。生物还原剂，如 N-乙酰半胱氨酸、MESNA 或半胱胺，增加了还原型同型半胱氨酸的可用性，从而促进了其随后基于 CBS 的消除。在 HCU 的转基因 I278T 小鼠模型中，生物还原剂的给药显着增加了血浆中蛋白质未结合的同型半胱氨酸的比例，这提高了共同给药的基于 CBS 的 ERT 的疗效，血浆总同型半胱氨酸水平显着降低证明了这一点。这些结果阐明了基于 CBS 的 ERT 的作用机制，并揭示了进一步提高其疗效的新型药理学方法。